Nonmuscle myosin II-B is almost ubiquitously expressed in mammalian cells, but the functions of the protein are incompletely understood. Previous work from this laboratory has demonstrated association of nonmuscle myosin II-B (NMII-B) with the plasma membrane in Xenopus (J. Cell Biol. 134: 675-687, 1996). In Drosophila, myosin II has been shown to interact with the gene product (Dlgl) of the tumor suppressor gene l(2)gl, a plasma membrane protein whose absence is associated with tumors in neuronal tissues, and the association is regulated by a kinase which binds to Dlgl (Kalmes et al., J. Cell Sci. 109: 1359-1368, 1996). To determine if similar localization mechanisms operate in mammalian cells, plasma membranes were purified from rat pheochromocytoma PC12 cells. A fraction of NMII-B was tightly associated with the membranes, but was released in a time- and dose-dependent manner by incubation with ATP-Mg. Release of NMII-B was potentiated by phosphatase inhibitors. Immunoprecipitates of NMII-B from PC12 cell lysates contained kinase activity which phosphorylated a GST fusion protein containing the putative phosphorylation site of Mgl, the mouse homolog of Dlgl. NMII-B immunoprecipitates also contained a protein of approximately 115 kD which was phosphorylated and released on incubation with ATP. When GST- Mgl fusion protein was incubated with PC12 lysates, precipitated with glutathione-agarose and subjected to in vitro kinase assay, the precipitates were found to contain kinase activity which phosphorylated both GST-Mgl and an associated approximately 115 kD protein. Since Dlgl forms heterodimers, it is proposed that the approximately 115 kD phosphoprotein is the native rat Dlgl homolog. GST-Mgl may bind kinase directly, or through native Mgl. These results suggest that binding of NMII-B to the plasma membrane in PC12 cells may occur through interaction with the mammalian homolog of Dlgl, and that the binding is inhibited by phosphorylation of the rat Dlgl homolog. Work is now underway to identify and characterize the kinase activity which binds and phosphorylates mammalian Dlgl homologs.